Functional studies to elucidate mechanistic interactions between different bacterial species within the human microbiome are critically needed to understand their role in this microbial community. Research suggests that non-diphtheriae Corynebacterium species play a significant role in structuring healthy nasal and skin microbiota. These generally harmless commensals are implicated in affecting colonization by Staphylococcus aureus and Streptococcus pneumoniae, pathogens that also frequently colonize nasal/skin sites. Our data indicate that some Corynebacterium species hydrolyze analogs of human skin-surface lipids releasing free fatty acids (FFAs) with anti-pneumococcal activity. Thus, we hypothesize that Corynebacterium spp. modify their habitat by producing lipases that release FFAs with antibacterial activity from host epithelial-surface lipids and that the level of these lipid substrates impacts the composition of the skin microbiota, diminishing pathogen colonization. The overall objective of this proposal is to gain genetic and mechanistic knowledge of how commensal Corynebacterium spp. release these antibacterial FFAs from host surface lipids and to determine if altering skin lipid composition affects microbiota composition. The rationale for this study is that uncovering these mechanisms will create opportunities to identify specific strains, genes and/or Corynebacterium-produced compounds with potential for therapeutic use. We will use a combination of culture-based approaches and human samples to identify relevant genes, and the impact of their expression, in culture and on epithelial surfaces. The Specific Aims of this proposal are (1) to determine how Corynebacterium spp. release antibacterial free fatty acids from host lipids, (2) to identify and characterize the regulatory mechanisms that control activity of extracellular lipases in lipid-requiring and lipid-independent Corynebacterium spp. and (3) to determine the impact of a topically applied skin-surface-lipid analog on the microbiota of three different human skin sites (dry, moist and oily). To accomplish these, we will use a combination of genetic, biochemical and bioinformatic approaches (Aims 1 and 2), along with a 16S rRNA gene-based approach (Aim 3). The significance of this work will be to provide the foundational mechanistic information that is critically needed to identify specifc strains of Corynebacterium spp. and Corynebacterium-produced compounds with potential as alternative, non-antibiotic therapies to prevent infections by pathogens, such as S. pneumoniae and S. aureus. The three main innovations in our strategy are (1) using functional molecular studies of bacteria in coculture to elucidate the role of different bacterial species within the human microbiome, (2) choosing a system that allows for non-invasive studies directly in the human host, and (3) translating our findings to test a simple intervention on human skin surfaces. Our long-term goal is to develop new and sustainable ways to manage the composition of nasal and skin microbiota to prevent disease by excluding or controlling pathogens.